inaccessible to bulk methods and relevant to
many interactions between proteins and ligands.

The use of an inherently nonequilibrium method to obtain equilibrium binding energies also
grants access to molecular interactions that equilibrate over very long time scales (e.g., nucleosome assembly) and that can only be currently
measured by indirect techniques such as competition assays (2, 22, 34). The FTLB relates work
measurements to binding energies without making any assumption on reaction kinetics or the
ideal solution limit. Therefore, it might be also
used to test the explicit breakdown of the law of
mass action in conditions where it is not applicable, for instance, in crowded environments, where
ligands exhibit compartmentalized dynamics due
to steric hindrance interactions (35). Lastly, the
applicability of the FTLB is not restricted to biomolecular reactions and might be directly applied
to other interacting systems that can only be explored through nonequilibrium methods.

MagReps (grant 267 862), Framework Programme 7 Infernos(grant 308850), the Catalan Institution for Research and AdvancedStudies (ICREA) Academia 2013, and Ministerio de Economia yCompetitividad (grant FIS2013-47796-P). All data used in this studyare included in the main text and in the supplementary materials.

Nucleosome assembly during S phase is tight- ly coupled to DNA replication (1). The initial step of replication-coupled (RC) nucleo- some assembly is the deposition of histone H3-H4 onto replicating DNA, which is fol-lowed by the rapid deposition of histone H2A-H2B (2, 3). Deposition of new histone H3-H4requires the action of histone chaperones (4).Replication protein A (RPA), a complex that inyeast is composed of the Rfa1, Rfa2, and Rfa3subunits, binds single-stranded DNA (ssDNA)at replication forks after double-stranded DNA(dsDNA) is unwound by the replicative helicaseminichromosome maintenance (MCM), facilitatesthe movement of the replisome, and functionsas a “unique harbor and binding platform” duringDNA transactions (5–8). We studied the potentialroles of RPA in RC nucleosome assembly.

RPA copurifies with both histones H3 and H4
(17). We confirmed this interaction and showed
that the RPA–H3-H4 interaction was not mediated by DNA (Fig. 1C). In vitro binding assays
demonstrated that recombinant RPA directly
binds free H3-H4, but not (mono)nucleosomal